PT101027A - Process for their preparation and their use as pharmaceutical compositions - Google Patents

Description

The present invention relates to peptides having pharmaceutical utility, processes for their production, pharmaceutical compositions containing them and their use as pharmaceuticals.

More particularly the present invention provides a compound of the formula I in which R1 is hydrogen, an amine or benzyloxy protecting group optionally substituted on the n ring is 0 or A is a direct bond, Val, Leu, Ala, Ile, trimethylsilyl-Ala or a divalent radical of formula (a) wherein R 1, R 2,

(a) wherein ring A is optionally substituted by hydroxy or C1-4 alkoxy. A4 is a direct bond or divalent radical of formula (b)

R1 is hydrogen or C1-4 alkyl and Y1 is the residue attached to the α-amino acid α-carbon and -CH2-CH2 -N (C1 -4 alkyl) 2'-pyridazin-2-yl- methyl, benzimidazol-2-yl methyl, 1H-1,2,4-triazol-3-yl-methyl, pyrazol-3-yl-methyl, indazol-3-yl-methyl or a radical of formula (c) or (d) optionally protected

(c) wherein R 2 and R 3 are independently hydrogen, halogen, C 1-6 alkyl or trityl, at the most R2 or R3 is H and R4 and R6 are independently hydrogen, C1-4 alkyl, hydroxyl, 4, CF 3, phenyl or halogen, at the most R 4 or R 6 is H, or A and A 4 together form a radical of the formula (aa)

(Wherein R3 is as defined above and R1a and R3 form together) (Cl2) and m is 2, 3, 4 or 5 and 1) X is a divalent radical of formula (e)

X2 attached by an X1 is O or S and a radical of which R3 is H or C1 -C4 alkyl, and A5 is hydrogen; CF3; a radical, wherein X is any direct bond or an α-amino acid residue and Y 2 is NH 2, C 1-4 alkylamino, di- (C 1 -C 4 -alkyl) amino or a nitrogen-containing heterocyclic radical to Z 2; a -CH 2 radical; wherein Y 3 is heteroaryl; a -CH 2 -Y 3 radical; or formula (k) to (m) j

5 X

-CH2 -CH2 -CH2 -CH2 -CH2 -CH2 -CH

Rn (m)

(D-R9-x10) -CH2 -CH2-X2 wherein Y4 is tri- (C1-4 alkyl) methyl or a residue

Ring B is pyridyl. . ·. R 1 and R 2 are independently C 1 -C 4 alkyl, C 1 -C 4 alkoxy, CF 2, halogen, nitrogen or cyano and R 2, R 3 and R 4 are independently selected from the group consisting of: R 11 and R 11 are independently nitrogen, cyano, CF 3, carbamoyl, CO 2 R 12 '-CH = CH-CH 2 and R 2 is also a divalent radical of formula

to kyl alkyl at < yl (e2) W · 6

when A 2 is H or (2) X is a divalent radical of formula (f)

(f) and is -Z -Z -Y "or a radical of formula (k) to (o) as defined above or OR 13 or wherein R 13 is C 1-6 alkyl optionally substituted by OH or. is interrupted by O and R4 and R4 are independently independently hydrogen, C1-6 alkyl, C5-7 cycloalkyl or benzyl, or (3) X is a divalent radical of formula (g)

and Ac is -Ζ, -Ζ-Y as defined above or D 1 Z Δ 4) X is a divalent radical of formula (h) or (j)

(h) (j) " W '7 7

and A 2 is a radical of the formula (k) a (O), -CH 2 -Y 3 or -CH 2 -X 1 -Y 3 as defined above, with the proviso that only or A 2 can be a direct bond when n is 0 and A 3 or A4 is not a direct bond when n is 1, and its physiologically hydrolyzable and acceptable esters or amides, in free form, in salt form or in the form of complexes.

Examples of protecting groups R are described e.g. in " Protective Groups in Organic Synthesis, ', T.W. Greene, J. Wiley & Sons NY (1981), 219-287, for example acyl such as acetyl, methoxysuccinyl, hydroxysuccinyl or benzoyl optionally substituted on the phenyl ring with e.g. p-methoxycarbonyl, p-methoxy or p-nitro; alkoxycarbonyl such as t-butyloxycarbonyl; arylmethoxycarbonyl such as 9-fluorophenylmethoxycarbonyl or benzyloxycarbonyl optionally substituted on the phenyl ring with p-methoxy, p-nitro, p-chloro or m-phenyl; arylmethyl such as benzyl optionally substituted on the ring with p-methoxy, p-nitro or p-chloro; or arylsulfonyl such as phenylsulfonyl optionally substituted on the ring with p-methyl or p -methoxy or naphthylsulfonyl optionally substituted on the ring with e.g. amine or di (C 1-4 alkyl) amine.

When R is benzyloxy, it is preferably hydroxy-substituted benzyloxy or C1-4 alkoxy < Preferably R is unsubstituted benzyloxy.

Halogen is preferably fluorine or chlorine.

When A3 is a substituted radical of formula (a) it is preferably substituted by C1-4 alkoxy, preferably to -C = O. δ

By α-amino acid is meant a natural or commercial or unnatural α-amino acid or an optical isomer thereof. An unnatural α-amino acid is an α-amino acid that is not incorporated into a protein under the mRNA direction, e.g. β-Nal, a fluoro-α-amino acid such as fluoroalanine, trimethylsilyl-Ala or an α-amino acid such as

(CH 2 n 2 HN 55. nh

2 ΚΗΛι

Wherein n is an integer from 1 to 6 and n is an integer from 1 to 12.

Protecting groups which may be present in are protecting groups of O, S or N function on the amino groups of the side chains of an α-amino acid. The N-protecting groups are eg as described above for R or C 3-8 alkyl such as isopropyl, formyl, a sugar residue such as 1-deo-fructosyl or α-glucosyl (1-4) -deoxyfructosyl, dihydroxy- C3-6 -alkyl such as dihydroxypropyl, C5-6 cycloalkyl, such as cyclohexyl or tropinyl. O and S protecting groups for the hydroxyl and thiol functions are known and may be e.g. methyl, t-butyl or benzyl.

When it is a heterocyclic radical, it may be e.g. a 5- or 6-membered ring, e.g. piperidino or pyrrolidinyl. 9

Examples of heteroaryl such as Y 3 include eg 5-, 6- or 7-membered unsaturated heteroacid radicals comprising at least one nitrogen and optionally further heteroatoms such as N, O or S. It is preferably heteroaryl comprising from 1 to 4 nitrogen atoms, eg pyridyl , triazolyl, tetrazolyl, triazin-dionyl.

In ring B of the radical (o), the nitrogen atom may be in o, m or para. When ring C in the radical (k) is pyridyl it may be 3-, 4- or 5-pyridyl.

The radicals (e), (e2), (f), (g) and (j) are derived from Asp and comprise an asymmetric carbon atom and the radical (h) comprises two asymmetric carbon atoms and thus lead to optical isomerism. It will be understood that the present invention includes all the individual isomeric forms and diastereoisomers as well as mixtures, eg racemates, unless otherwise stated. The radical of the formula (e) attached to which is hydrogen may exist in cyclic form and not cyclic eg as follows: NR, co2h

OH

It should be understood that whenever tautomeric forms occur, the present invention encompasses lactol and oxo carboxylic acid forms, ie although compounds of formula I wherein X is a radical of the formula (e) are defined by Convenient by reference only to the form of oxo carboxylic acid, the invention is not to be understood as being in any way limited by the particular nomenclature or graphical representation employed. Similar considerations apply to the starting materials which show lactol / oxo-carboxylic acid as described hereinafter.

The same considerations apply to the radical of formula (h) which may exist in linear form and cyclic form as follows:

OH

-NR6) The base / ag H +

and to compounds of formula I comprising a radical of formula (h) and the corresponding starting materials.

By the term " physiologically hydrolyzable and acceptable esters or amides " is intended to mean esters and amides which are hydrolyzable under physiological conditions to give alcohols or amines which are themselves physiologically acceptable, i.e. which are non-toxic at the intended dosage levels. as

Such esters or amides are obtained by esterification or amidation, respectively, of a compound of formula I wherein X is a carboxyl-bearing radical. Such esters include esters with an aliphatic or alicyclic alcohol or polyol having 1 to 12 carbon atoms. Such amides include amides with aliphatic amines, e.g. C1-4 alkylaminoalkylC1-4 alkylamine, or β-methoxyethylamine or aniline. 11

The compounds of the formula I may exist e.g. in the free form, the acid addition salt form or in the form of complexes. The acid addition salts may be formed with e.g. organic acids, polymeric acids and inorganic acids. Such acid addition salt forms include e.g. hydrochlorides and acetates. The salt forms may also include those obtained with the carboxyl group present in compounds of the formula I, e.g. alkali metal salts such as sodium or potassium salts or substituted or unsubstituted ammonium salts. Complexes are e.g. formed from compounds of formula I when adding organic substances, e.g. inorganic salts or hydroxides such as Ca and Zn salts, and / or an addition of polymeric organic substances.

In the compounds of formula I, the significances are preferred individually or in any combination or subcombination: 1. R is an amine or benzyloxy protecting group, preferably an amine protecting group. R is preferably benzyloxy when n is 0 and is a radical of formula (a). 2. A3 and A4 are not a direct link. 3. n is 0. 4. A 3 is a radical of formula (a) when n is 0. 5. A 3 is a direct bond, Val, Leu, Ala, Ile or trimethylsilyl-Ala. 6. R 1 is hydrogen or methyl, preferably hydrogen. 7. Y 1 is the residue which binds to the α-amino acid selected from Ala, Leu, His, Fen, Met, Trp, trimethylsilyl-Ala and Arg, Orn and Lys optionally protected in the side chain, or Y 1 is a radical of formula (c). (A) and (A) may also together form a radical of the formula (aa) when n is 0. 9. m is 2 or 3. 10. X is a radical of the formula other than H. 11. X is a radical of formula (g). 12. X is a radical of formula (h) or (j). 13. R 6 is hydrogen or methyl, preferably hydrogen. 14. Z- in Ac is the residue of a natural α-amino acid, preferably of an aromatic / heterocyclic α-amino acid, particularly Pro. 15. Zn in Aβ is the residue of a natural α-amino acid, D is preferably an aliphatic α-amino acid, particularly an aliphatic α-amino acid having no other functional group, more preferably Val. 16. 17. X is a radical of formula (e) optionally esterified or amidated and Ag is a radical of formula (k), (1) or (o). X is a radical of the formula (e), (h) or (j) optionally esterified or amidated and Aj is -C1 -X2 -Y2, preferably 17.

-ch2-s-y3 or -ch2-y3. 18. X is a radical of formula (h) or (j) optionally esterified or amidated and A 1 is (k). 19. X is a radical of the formula (j) optionally esterified or amidated and A5 is a radical of the formula (m). 20. The radical of the formula (m) is mono-substituted, preferably in para, more preferably 4-nitrophenyl. 21. All α-amino acid residues present except X have the L configuration. X has the D or L configuration. The asymmetric carbon in (h) bearing the OH group has the S configuration. Preferably the asymmetric carbon in ) bearing Rg has the R.

In a series of specific embodiments, the present invention also provides a compound of formula I wherein n is 0, A3 is a direct bond, Val, Leu, Ala, Ile or trimethylsilyl-Ala, A4 is as defined above, or A3 and A4 together form a radical of the formula (e1) or (e2) as defined above and i) X is a radical of the formula (e2 or (e2) as defined above and Ar is H or

(Ii) X is a radical of the formula (e) or (f) and A5 is as defined above or a radical of the formula (k), (1) or (m) wherein X is O, of formula (g) and A5 is -Z1 -Z2 -Y2 or iv) X is a radical of formula (j) and A1 is a radical of formula (k), (1) or (m) wherein X1 is 0. 14

The present invention also provides a process for the production of a compound of formula I, which process is characterized by: a) removing at least one protecting group from a compound of formula I in protected form or adding a protective group R to N-terminal group of a compound of formula I; or b) converting a compound of formula I into another compound of formula I; or c) coupling via an amide bond of two peptide fragments, each of which contains at least one amino acid in protected or deprotected form and a peptide fragment containing a radical of formula (e) to (j) as defined above, the peptide fragments being such as to provide a protected or deprotected peptide having the sequence according to formula I above and if necessary removing the protecting group (s) from a compound of formula I in protected form; or

d) for the production of a compound of the formula I wherein X is a radical of formula (e) or (h) and A5 is a radical of formula (k), (1) or (o) or -CH2-X1 A compound of the formula III in which R1, R2 and R3 are as defined above, X 'is a reaction of a compound of the formula III wherein R1, a radical of the formula (e) or (h) and Z is a leaving group, X is halogen,

with a corresponding phenol, thiophenol or HX3 pyridine or an acid of the formula ΗΧ, -CO-Y. or a functional derivative 1-4 or ΗΧΥ-Υ^; or

e) for the production of a compound of the formula wherein R 1, R 2, R 3 and R 4 are as defined above and R 1, R 2, R 3, R 4, is a ^ 15 16

aliphatic or alicyclic residue, oxidation of a compound of formula V

(A) -A] -A] -A3-A4-Asp (V)

Wherein R1, R2, R3 and R4 are as defined above and recovering a compound of formula I thus obtained in the free or salt form or in the form of a complex.

The processes (a) to (e) above may be carried out according to known conventional techniques. Removal of a protecting group in process step (a) may also include the removal of R in the N-terminal group from a compound of formula I. For example, when R is 16 x V 'benzyloxycarbonyl, this group can be removed by hydrogenation in the presence of a catalyst eg Pd.

According to process step (b), for example, for the production of a compound of formula I in which X comprises a carboxyl group, a compound of formula I wherein X comprises a carboxyl group which is esterified or amidated may be hydrolyzed. Such hydrolysis may be effected by treatment with a suitable base or by acid hydrolysis, for example in the presence of trifluoroacetic acid.

Further, according to process step (b) for the production of a compound of the formula I in which X comprises an esterified or amidated carboxyl group, a compound of the formula I wherein X may comprise a carboxyl group or an esterified carboxylated group. Such ester or amidation formation may be carried out using any of the known techniques, for example conversion of the carboxyl group into a functional reactive group, eg a corresponding halide or anhydride, or using a compound of formula I wherein X is a radical of the formula h) as lactone and reacting such a group with the selected alcohol or amine.

According to an embodiment of process step (b), for the production of a compound of formula I wherein X is a radical of formula (g), a compound of formula I wherein X is a radical of formula (e) or (e2) and A2. and H may be reacted with a compound of the formula II (II)

in which it is as defined above. This process can be carried out in analogy to the known techniques used in the preparation of semicarbazones.

A compound of formula I wherein X is a radical of formula (e) may also be converted according to known techniques into a compound of formula I wherein X is a radical of formula (e2) and vice versa. Process step (c) may be carried out by known techniques of peptide chemistry. A peptide fragment comprising a radical of the formula (e1) to (j) is meant the carrier moiety itself of the protecting group in the -NR- moiety and a suitable end, e.g. A or CH- L · cl side. Process step (d) may conveniently be carried out using a Dess-Martin reagent, e.g. in the presence of a base or a silver salt which precipitates with halogens or according to the Swern oxidation processes.

Where desired, in these reactions protective groups may be used for functional groups which do not participate in the reaction.

These may be for example amine protecting groups, carboxyl protecting groups, acetal groups, etc. When the desired reaction is complete, the protecting groups can then be removed.

Each of the foregoing processes may be carried out using starting materials in the form of one or other of the individual optical isomers or in the form of mixtures [related to the asymmetric carbons 18 present in the radicals of the formulas (e1) to (j) as X or such radical precursor]. For convenience the starting materials are used with S or R enantiomers to produce a compound of formula I wherein the asymmetric carbon in the radicals of the formulas (and a) (j) has the S or R configuration, respectively.

The starting materials used in steps (d) or (e) of the process may be prepared in analogy to process step (c). Since the production of the starting materials is not particularly described, the compounds are known or can be prepared in a manner analogous to known methods practiced in this field.

The following examples serve to illustrate the invention. All temperatures are in ° C.

The following abbreviations were used: THF = tetrahydrofuran TFA = trifluoroacetic acid MeOH = methanol EtOAc = ethyl acetate DCC = dicyclohexylcarbodiimide HOBT = hydroxybenzotriazole Z benzyloxycarbonyl t.a. = ambient temperature Fmoc = 9-fluorenylmethoxycarbonyl a = amorphous 19

EXAMPLE 1 Z-Val-Met-Aspartic Acid Z-Val-Met-Asp (OH) -HO ester of 1.alpha.-Val-Met-aspartic acid aldehyde dimethylacetal-6-tert.butyl (1.17 g) was resuspended in CH 2 Cl 2 (150 ml), and TFA (15 ml) and water (1 ml) were added and stirred for 2.5 hours at rt. The solvent was evaporated, added twice and again evaporated. The crystalline residue was dissolved in water (60 ml) with heating, decanted to remove some of the undissolved material and crystallized at 5øC, giving the first crop of the product: mp 117øC. All mother liquors were collected and chromatographed (SiC> 2, acetone / hexane / EtOAc 60/40 / 0.5) to give a second crop of product, crystallizing on titration with water.

EXAMPLE 2: Semicarbazone Z-Val-Met-Asp (OH) -H The compound of Example 1 (0.27 g, 0.55 mmol) in MeOH (2 mL) was combined with semicarbazide, HCl (0.6 mL a 1 molar solution), and then 5 drops of pyridine were added. the product crystallizes after 10 minutes at rt: mp 233-235 ° C. i.

EXAMPLE 3 The compound of Example 1 (0.48 g, 1 mmol) was dissolved in a mixture of MeOH (2 mL) ) water (0.5 ml) and 3 drops of pyridine. N- (hydrazinocarbonyl) -Pro-Val-N (CH3) 2 (0.3 g, 1 mmol) was dissolved in MeOH (1 mL), water (2 mL) and 3 drops of 2N HCl. Both solutions were combined and heated for 2 minutes at 40-50 ° C. The product crystallizes upon cooling: mp 118-120 ° C.

By repeating the procedure of example 1, using the corresponding starting materials, the following compounds may be prepared:

EXAMPLE 4 Z-ValFen-Asp (OH) -H

EXAMPLE 5 Z-Val-His-Asp (OH) -H

By repeating the procedure of Example 2 or 3, respectively, using the corresponding starting materials, the following compounds of the formula IA can be prepared: Z-A3-A4-n

(IA) wherein A 3, A 5 and A 5 are as defined above 21

Example A3 A4 A5 mp 6 C Val Fen H a 7 Val His H 150 8 Val Fen Pro-Val-N (CH 3) 2 126-129 9 His Pro-Val-N (CH3) 2 Ala-Tyr-Val (3S, 4RS) -3-Amino-3- (2,6-dimethylbenzoyloxy) -4-oxopentanoic acid tert.-butyl ester, (0.6 mmol) was treated with the Dess-Martin reagent (0.77 mmol) in CH 2 Cl 2 (4 mL) ) for 45 minutes and filtered. Acetone and 0.5 N NaOH were then added and the acetone was evaporated. The residual crystalline material was washed with H2 O acidified with 10% tartaric acid and extracted with EtOAc. The combined EtOAc extracts gave, after evaporation, the ester t. butyl the product as yellow crystals. These were dissolved in CH 2 Cl 2 (4 mL), TFA (4 mL) was added and the mixture was stirred at r.t. for 15 minutes. The mixture was evaporated to dryness and chromatographed (SiO 2, acetonamide / hexane / EtOAc 60/40/1) to give the product as slightly colored crystals. EXAMPLE 12 (Z-Valyl-alanyl) - (3R, 4S) -3-amino-4-hydroxy-5- (2,6-dichlorobenzoyloxy) 3-amino-4,5-dihydroxypentanoic acid and 4-dimethylaminopyridine were dissolved in pyridine and 2,6-dichlorobenzoyl chloride was added dropwise. The reaction mixture was stirred overnight at ambient temperature, then ice and water were added and the mixture was extracted with AcOEt. The organic phase was washed with water, then with NaCl solution, dried over Na2SO4, filtered, evaporated and the crude product was chromatographed to give the title compound. The 3R, 4S derivative of the title compound can be prepared as follows: Z-Val-Ala-OH (0.26 mmol), DCC (53 mg, 0.26 mmol) and HBOT (20 mg, , 26 mmol) were dissolved in THF / DMF (2 mL / 2 mL) and stirred for 5 minutes before (3S, 4RS) -3-amino-4-hydroxy-5-2- , 6-dimethyl-benzoyloxy) -pentanoic acid (87 mg, 0.26 mmol) in THF (2 mL). The reaction mixture was stirred overnight at rt, evaporated and chromatographed (SiC> 2, EtOAc / MeOH / NH3 95/5/5) to give the title compound (3R, 4S). EXAMPLE 13 (3-Amino-4-oxo-5- After 15 minutes a solution of the compound of Example 12 in CH2 Cl2 was added dropwise and the reaction stirred for 1 hour at -48 DEG C. Triethylamine was added and stirring was continued for 3 hours at The organic phase was washed with NaHC03 solution and NaCl solution, dried over Na2SO4, filtered and evaporated.The residue was chromatographed giving the title compound.

By repeating the process of Examples 1-3 and 11-13, using the corresponding starting materials, compounds of formula IB

O·.·

R (IB) wherein A 3, A4, R 5 and R 8 are as defined below,

Cu CM 'w-' cd

The compound according to claim 1, wherein the compound of formula (I) Γ -I i-1 • * H • tH mH * H rH rH Ή * HO O OO * H • HOO o 'NNMNQONNNC cc CNNC a rl e 0 <u 0 0 C3 c 0 0 • H ω X3 Λ JC J3 0 0 • Q xs C l 1 1 1 -O J3 1 1 0 1 n rN mm | | 0 0 r-1 Cu O Cu Cu -H rH X 2: Çh 33 O O r O • rH • rH I • H • H • rH • rH • rH • rH • rH Π3 1 | rsj Q T3 I T3 I T3 1 Ό I Ό TJ Ί3 vo 2 1 & 1 vO 1 vO I vO 1 M3 1 v £> 1, p., P. CM CM CM CM CM CM CM CM CM CM CM> 33 33 33 33 33 33 33 33 33 33

I

CM &lt; uw &quot; CM &quot; &quot; CM &quot; &quot; 7 &quot; ON &quot; Χ χ χ χ χ χ χ Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή Ή (A z z Ή Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π Π, Ky T3 | 1 * sO 1 Ό 1 Ό 1 Ό 1 w W CM CM CM CM e e XXX

O

Ot s ω x ω

m co co σ \ CM CS CM CM CM o co m

- - - - - - - - - - - - - - - - - - - - - ο 0) • Σ. U [Ιο a w- 04ο

ÍK

Xos

&gt; OXcuS ωx Cd

The compound according to any one of claims 1 to 4, wherein R 1 and R 2 are independently selected from the group consisting of: • H • H • H • H • H • H • H • H • H • H • H • O • O • O • O • O • HO • O • NNNNNNNNN o NNNNNNCC C3 C ac C cc N c CCCCC <u <D <U <U; CU a; ω to ω ω <and <U ω cu x X X X J2 X X X -Q a; -D X * x &gt; 1-yl) -1H-indol-1-yl] - rH • rH • rH • rH • rH • rH • rH • rH • H • H • HO • rH • rH • rH • rH • • • • • • • • • • • • • • • • rH Ό Ό Ό rH Ό Ό Ό ~ v T3 -n rH Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό Ό In this case, you will see that you are in the same position as in the case of a CM CM CM CM, CS CM CM CM CS CS CS CS CS CS CS CS CS CS CS CM

X H 03 rs r-J O cn CS ♦ H rj o &gt; SC or N as SC • H c o m ιΛ m *> O Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ

Π3 03 c M oj co C co co co C co Co C co co c co c u c cd M rH • rH • H • rH • rH • rH rH • D • H • H • H • rH> , • rH rH rH OX <2 s SC XXX < X X X 1 X X X 1 X 1 < &lt; CS υ Z

Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z &lt; tb &gt; &gt; &gt; &gt; &gt; &gt; &gt; &gt; &gt; &gt; EXAMPLE A3 (R) 1a) 20 c (MeOH) 2-Hydroxy-

C

O or O N C <j

u * »h Ό I Ό cM

0 N C ω J3 1 rH CJ Ό í vO CNJ

in ΙΛ in tn ΓΝ rN N O O O O Γ Γ0 rH < &lt; V)

O-H cd ctj r-l I- \ < &lt;

The rH m m nm nm m

TriMe-silyl-Ala-H-2,6-diCl-benzoyl-1aAm m VO mm m 28

EXAMPLE A3 A4 Ry Rx M20 c (MeOH) EXAMPLE 58:

Cl

Z-His-Asp-CH 2 -O-CO- (O

Cl 20

[Î ±] D = -20.3Â ° C = 1 in MeOH EXAMPLE 59:

Cl

Fmoc-Val-His-Asp-O-CO- (Q

Cl EXAMPLE 60:

C6 H5 -CH2 -O, CO-His-Asp-CH2-O-C0

Cl [Î ±] 20D = -21.5Â ° C c = 1.07 MeOH EXAMPLE 61:

Cl

C? H? -CH? O

CO-His-Asp-CH 2 -O-CO- (Q

(i.e.

Cl EXAMPLE 62:

Cl.fe> S02-Val-Ala-Asp-CH2-O-C0-

Cl

By repeating the procedure of Examples 1-3 and 12-13 and using the corresponding starting materials, the following compounds of formula IC can be prepared

OH Z-Val-A4-NH

0

IC

R x

wherein A-, R3 and R4 'are as defined below.

EXAMPLE 4 Ala H 2,6-diCl-benzoyl R, S 64 Ala C 2 H 5 Cl -CO 3 R 6 Cl 65 Ala -44 2,6-diCl-benzoyl R, S 66 Ala -OCH2-OCH3 2,6-diCl-benzoyl R, S EXAMPLE 67; (3RS) -3- (Z-Valyl-phenylalanylamino-4-oxo-6-p-nitrophenyl) -trans-hex-5-enoic acid tert.butyl ester tert.butyl- - (Z-valyl-phenylalanyl) amino-4-oxo-pentanoic acid (0.2 g, 0.29 mM) was dissolved in THF (15 mL) at 5 DEG C. NaH (25 mg, 0.59 (0.18 g, 1.1 mM) in THF (2 ml) was added at 0 ° C and the reaction mixture was stirred for 45 minutes at this temperature The reaction mixture was poured into 5% tartaric acid, extracted with ethyl acetate, the organic phases dried over Na 2 SO 4, evaporated and the product purified by chromatography to give the title compound. (0.8 g) was dissolved in MeOH (25 ml) containing 8% HCl, left at room temperature overnight, evaporated to dryness and the residual crystals washed with ether to give the title compound in form of white crystals, mp 168-171 ° C. The starting materials can be prepared as follows: EXAMPLE 69 (3S) -3- (Fluorenylmethoxycarbonyl) amino-5-iodo-4-oxobutanoic acid tert.-

Triethylamine (1.3 ml, 9.6 mmol) followed by ethyl chloroformate (0.92 ml, 9.6 mmol) was added to (3S) -3- (fluorenylmethoxycarbonyl) amino-3- butyl alcohol (3.5 g, 8.5 mmol) in THF (60 mL) at -10 ° C. After 10 minutes, the diazomethane solution in ether was slowly added and the reaction stirred for 45 minutes at 0-5 ° C. HCl in ether (2N) was added at 5-10 ° C until gas evolution ceased. The reaction mixture was evaporated to dryness, resuspended in acetone (50 ml) and added Nal (4 g) and stirred for 1 hour at r.t. Ether (150 ml) was added, the reaction mixture was filtered and evaporated. The residue was chromatographed (SiO2, EtOAc / hexane) to give the title compound as slightly yellow crystals. EXAMPLE 70 (3S) -3- (Fluorenylmethoxycarbonyl) amino-5- (2,6-dimethylbenzoyloxy) -4-oxopentenoic acid tert.butyl ester 3S-3- (fluorenylmethoxycarbonyl) amino acid tert.- -5- (2,6-dimethylbenzoyloxy) -4-oxobutanoic acid (1 g, 1.8 mmol), 2,6-dimethylbenzoic acid (0.5 g, 3.3 mmol) and AgOAc (0.6 g, 3.6 mmol) were dissolved in acetone (25 mL) and refluxed for 1 hr. After filtration and evaporation the crude product was chromatographed (SiO2, ether / hexane 3/7) to give the title compound. EXAMPLE 71 (3S) -3-Fluorenylmethoxycarbonyl) amino-5- (4-nitrophenoxy) -4-oxopentanoic acid tert -butyl ester 3S-3- (Fluorenylmethoxycarbonyl) amino-5-iodo-4- oxo-butanoic acid (1 g, 1.8 mmol), p-nitrophenol (0.5 g, 3.7 mmol) and K 2 CO 3 (0.38 g, 2.8 mmol) were refluxed in acetone ml) over 30 minutes, CH 2 Cl 2 was added and the organic phase washed with NaHCO &gt; The organic phases were dried, evaporated and chromatographed (silica, 2x EtOAc / hexane 2/8) to give the title compound as a yellow oil. EXAMPLE 72 (3R, 4S) -3-Benzylamino-4,5- (isopropylidenedioxy) -4- (3R, 4S) -3-amino-4,5-dihydroxypentanoic acid ethyl ester pentanoate (Y. Yamada, Tetrahedron Leters 1983, 24, 3009) and 10% Pd / C in ethanol were stirred at room temperature for 30 minutes.

ν '. in H2 atmosphere &lt; Filtration and evaporation of the reaction mixture gives ethyl (3R, 4S) -3-amino-4,5- (isopro pydenedioxy) pentanoate which is used without further purification. b) Z-Val-Ala-OH was dissolved in THF, HBOT20 and DCC were added at 5 ° C. After stirring for 20 minutes at 5øC, diisopropylidenedioxy) pentanoate in THF was added. The reaction mixture was stirred overnight at ambient temperature, filtered, evaporated and chromatographed to give (Z-valylalanyl) - (3R, 4S) -3-amino-4,5- (isopropylidenedioxy) ) pentanoic acid. c) Compound 74b) was dissolved in AcOH / H2 O (75/25) and stirred at 40 ° C for 4 hours. After evaporation, water was added and the mixture was extracted with AcOEt. The combined extracts were washed with water, NaHCO 3 solution, NaCl solution, dried over Na 2 SO 4, filtered and evaporated to give the title compound.

The compounds of formula I, their hydrolysable and physically acceptable esters and amides (hereinafter referred to as compounds of the invention) have pharmaceutical activity and are therefore useful as pharmaceuticals.

In particular, the compounds of the invention inhibit IL-β3 secretion as indicated in the following in vitro test using THP-1 cells and in vivo test methods. 6 a) 900 μl of THP-1 cells (0.5 x 10 cells) along with 35 100 U gamma-interferon / 0.9 ml RPMI 1640 medium (containing 2 mM L-glutamine and 5% fetal serum of heat-inactivated vellum) were pipetted into 24-well tissue culture plates. 100 μl of the compound to be tested were then added. After 3 hours at 37øC in 5% Å / 95% air, 10æl lipopolysaccharide at 500μg / ml was added and the incubation continued for a further 40 hours. Appropriate controls (with or without stimulus, solvent) were also included. The media were then removed and clarified by centrifugation at 1000 g for 10 min. 1.0 ml of 0.01% digitonin was added to the wells to lyse the cells that are peeled off by scraping with a rubber scraper and left at 4øC for 10 minutes. The lactate dehydrogenase measurements were then performed immediately and the samples stored at -20 ° C until further determinations were made. The assays are: IL-β (medium and lysate), IL-6 (medium), TNF-α (medium), PGE 2 (medium and lysate), lactate dehydrogenase (LDH) and DNA (lysates). The IL-6β, IL-6 and TNF-α assays were determined using commercial ELISA kits (Cistron), PGE2 was measured using a standard RIA and the DNA determined fluorimetrically using DAPI.

In this test, the compounds of the invention selectively inhibit the release of IL-Ιβ in concentrations between about 0.01 and 100 μΜ. On the contrary the levels of IL-6, TNF-α, PGE2 and remain substantially unaffected and the compounds are non-toxic, since the LDH release is unaffected. For example, it has been determined that the compounds of examples 40 and 50 have an IC 50 value (concentration of compound which inhibits the release of IL-Ιβ by 50%) of 1 and 0.1 μΜ respectively.

Fever caused by LPS

A suspension of LPS. (Sigma, N2 L-5886, 100æg / 5ml glucose solution / Kg s.c.) was injected into male Tuttlingen SD (150-160g) male rats. Two hours later the body temperature was measured using a rectal probe attached to an ELLAB telethermometer. After 4 hours the test compound was given p.o. 2 hours later (6 hours after PLS administration) the temperature was again measured. The increase in temperature presented by the untreated controls is considered as 100% and in the treated group is expressed as a percentage of this value. ED 50 is the dose causing a 50% inhibition of the temperature rise determined in the control mice. In this test the compounds of the invention inhibit the increase in temperature caused by LPS when administered at a dosage in the range of 0.001 to 0.1 mg / kg p.o. It has for example been determined that the compounds of examples 40 and 50 have an ED50 value of 0.01 mg / kg p.o. and the compound of example 51 was ED50 of 0.05 mg / kg p.o.

Paw edema induced by carraqenane in rat

For each group 5 male OFA rats, 150-170 g weight, were used. The test compound was administered orally as a suspension in physiological serum / 0.5% tragacanth 1 hour prior to the injection of carrageenan. Carrageenan (0.1 ml of a 1% suspension in physiological saline) is given by subplantar injection into a hind paw. The paw swelling was measured with an antiphlogometer according to Kemper &amp; Arnelm. A control measure was taken immediately after the injection and swelling was measured after 3 and 5 hours. The mean reading value at 3 and 5 hours is determined after deduction of the reading from the control. The 37

values obtained from the treated animals are expressed as a percentage of the value obtained from untreated controls. ED 50 is the dose causing a 50% inhibition of carrageenan-induced swelling after 3 hours. In this test the compounds of the invention significantly inhibit carrageenan-induced swelling when administered p.o. at a dosage in the range of 0.02 to 5 mg / kg. It was determined, for example, that the compound of examples 40 and 50 have an ED50 value of 0.2 and 1 mg / kg p.o. respectively.

The compounds of the invention are therefore useful for the treatment of disorders with an etiology associated with or due to the excessive release of IL-β, eg in a wide variety of inflammatory conditions and diseases, for example calcium deficiency in tissues, degenerative processes in bone and cartilage, eg rheumatoid arthritis and osteoarthritis, inflammatory bowel diseases, irritative bowel diseases, septic shock, psoriasis, asthma, adult respiratory distress syndrome, type I diabetes, osteoporosis of various origin including eg climacteric or postmenopausal osteoporosis as well as osteoporosis due to old age, immobilization or trauma, arteriosclerosis and Alzheimer's disease.

For the above uses the dosage required will vary depending on the mode of administration, the particular condition being treated and the effect desired. However, in general satisfactory results can be achieved at daily dosages of between about 0.001 and about 100 mg / kg, preferably between 0.001 and about 10 mg / kg by weight. The daily dosages for larger, e.g. human, mammals are in the range of about 0.1 mg to about 1 g / day, for convenience given in one go, in dosages divided 2 to 4x / day or in the form of release controlled. 38

The compounds of Examples 50 and 51 are preferred.

According to the foregoing the invention also provides: a) A method for the treatment of disorders with an associated etiology or due to the excessive release of IL-β, eg as indicated above in a needy individual, which method is characterized by administering to said individual an effective amount of a compound) of formula I, a physiologically acceptable and hydrolyzable ester or amide thereof or a pharmaceutically acceptable salt thereof; b) A compound of formula I, a physiologically hydrolyzable and acceptable ester or amide thereof or a pharmacologically acceptable salt thereof for use as a drug, for example for use as an agent for example in the method described above.

The compounds of the invention may be administered by any conventional route, in particular nasally, enterally, preferably orally, e.g. in the form of tablets or capsules or parenterally e.g. in the form of injectable solutions or suspensions or in the form of suppositories. Dosage unit forms contain, for example, from about 25 μg to 500 mg of a compound of the invention.

J

The compounds of the invention may be administered in free form or in pharmaceutically acceptable salt form. Such salts may be prepared in conventional manner and have the same order of activity as the free compounds. 39

Further the present invention also provides: c) A pharmaceutical composition comprising a compound of formula I, a physiologically hydrolyzable and acceptable ester or amide thereof, as described hereinbefore, together with a pharmaceutically acceptable diluent or carrier. Such compositions may be produced in conventional manner. They may comprise up to 99.9% by weight of active ingredient.

Lisbon, 2 November 1992

J. PEREIRA DA CRUZ Agantt Qteial 4 «Wopriedafe industriai RUA VICTQR COWOOW, 10-A 3.» 1206 U3BOA

Claims (2)

1. A compound of the formula: (I) wherein R n is hydrogen, an optionally substituted amine or benzyloxy protecting group on the ring is 0 or 1, is Val, Leu, Ala, Ile or trimethylsilyl-Ala is Fen or Tir, is a direct bond, Leu, Ala, Ile, trimethylsilyl-Ala or a divalent radical of formula (a) (a) wherein ring A is optionally substituted by hydroxy or C 1-4 alkoxy, wherein A4 is a direct bond or a divalent radical of the formula (b) wherein R is hydrogen or C1-4 alkyl, and Y is the residue attached to the carbon atom α-amino acid and -CH 2 -CH 2 -N (C 1-4 alkyl) 2'-imidazol-2-yl methyl, benzimidazol-2-ylmethyl, 1H-1,2,4-triazol-3-yl 3-yl-methyl, indazol-3-yl-methyl, or a radical of the formula (c) or (d) R 2 and R 3 are independently hydrogen, halogen, C 1-4 alkyl, CF 3 or trityl, at most R2 or R3 is H and R4 are independently hydrogen, C1-4 alkyl, hydroxyl, C1-3 alkoxy, when at most R1 or R2 is phenyl or halogen, H or A3 and A4 form together a radical with the formula (aa) in which Y 1 is as defined above and R 2 and R 2 together form - (CH 2) m - (CH 2) n - 3, 13, 13 or 14, and i) X is a divalent radical of formula (e) (ei) co2h wherein Rc is H or C1-4 alkyl and Ag is hydrogen; CF3; a radical -Z2 -Z2 -Y2 wherein Y2 and X2 are each independently a direct bond or an α-amino acid residue, and Y2 is NH2, C1 -C4 alkylamino, di- (C1 -C4) alkyl) amino or a heterocyclic radical linked by a nitrogen to Z 2; a -CH2 -X2 -Y3 radical wherein is O or S and Y3 is heteroaryl; a -CH 2 -Y 3 radical; or a radical of the formula (3c) to (m) 00 K 9 Kn (m) (D B / (O) wherein Y4 is tri- (C1 -C4 alkyl) methyl or a residue having the ring B is pyridyl, the ring C is phenyl or pyridyl, R 'and Rg are each independently C C1-3 alkoxy, halogen, nitrogen or cyano, and R11, R11 and R11 are each independently nitrogen, and R1 is cyano, CF3, carbamoyl, CH2 CH2 CH2 CH3 or -CH = CHCO2 R12 wherein R12 is C1-6 alkyl, X being also a divalent radical of formula (e2) (e2) when it is H or (ii) X is a divalent radical of formula (f) and A5 is -Z-Z2 -Y2 or a radical of formula (k) a (o) as defined above or wherein R13 is C1-6 alkyl optionally substituted by OH or interrupted by 0 and 15. . R14 and R4 are each independently hydrogen, C1-12 alkyl, C5-7 cycloalkyl or benzyl, or iii) X 'is a divalent radical of formula (g) II (g) and Ac is -Z.-Z.-Y. as defined above or X is a divalent radical of formula (h) or (j). * \ and is a radical of the formula (k) a (o), -CH2 -Yg or -CH2 -X1 -Y3 as previously defined, provided that only A3 or A4 may be a direct bond when n is 0 and A3 or A4 is not a bond when n is 1; and their physiologically hydrolyzable and acceptable esters or amides, in free form, in salt form or in the form of complexes. A compound of the formula (I) according to claim 1, wherein n is 0, A 3 is a direct bond, Ala, Ile or trimethylsilyl-Ala is as defined in claim 1 or A and A together form a radical of the formula (aa) as defined in claim 1 and i) X is a radical of the formula or (ii) X is a radical of the formula (e) or (f) and Ac is -Z-Z-Y as defined in claim 1 or a radical of the formula (k), (1) or (m) as defined in claim 1 wherein X1 is 0, or iii) X is a radical of formula (g) and A3 is -Z-Z2-Y2 as defined in claim 1, or iv) X is a radical of the formula (j) and is a radical of the formula (k), (1) or (m) as defined in claim 1 wherein X1 is 0 and its physiologically hydrolyzable esters or amides and acceptable, in the free form, in salt form or in the form of complexes. The compound of formula (I) according to claim 1, wherein n is 0 and A 3 and A4 are not a direct bond and their physiologically hydrolysable and acceptable esters or amides in the free form in the salt form or in the form of complexes. 4®. A compound of the formula (I) according to claim 1 or 3, wherein the α-amino acid residue is selected from Ala, Leu, His, Fen, Met, Trp, trimethylsilyl -Ala and optionally Arg, Orn and Lys protected side chain, or a 9 9 (c) and their physiologically acceptable and hydrolyzable esters or amides, in free form, in the salt form or in the form of complexes. The compound of formula (I) according to claim 1, 3 or 4, wherein X is a radical of formula (e), (h) or (j), wherein A is other than H when X is (e) and their physiologically hydrolyzable esters or amides, in free form, in the form of salts or in the form of complexes. 6§. A compound selected from benzyloxycarbonyl-valyl-alanyl) -3R-3-amino-oxo-5- (2,6-dichlorobenzoyloxy) pentanoic acid ethyl ester, benzyloxycarbonyl-valylalanyl) -3-Amino-oxo-5- (2,6-dichlorobenzoyloxy) pentanoic acid, benzyloxycarbonyl-valylalanyl) -3R-3-amino-oxo-5- (2,6- (benzyloxycarbonyl-valyl-alanyl) -3R, S-3-amino-oxo-5- (2,6-dichlorobenzoyloxy) -pentanoic acid, (benzyloxycarbonyl- valyl-alanyl) -3R, S-3-amino-oxo-5- (2,6-dichlorobenzoyloxy) pentanoic acid, (benzyloxycarbonyl-valylalanyl) -3R, S-3-amino- oxo-5- (2,6-dichlorobenzoyloxy) pentanoic acid and benzyloxycarbonyl-valylalanyl) -3R-3-amino-oxo-5- (2,6-dichlorobenzoyloxy) pentanoic acid in free form, in the form of a salt or in the form of form of complexes. 7 ^. A process for producing a compound of the formula (I) and its physiologically acceptable and hydrolysable esters and amides, comprising: a) removing at least one protecting group from a compound of formula (I) in the form protected group or addition of a protecting group R as defined in claim 1 in the N-terminal group of a compound of formula (I); or b) converting a compound of formula I into another compound of formula (I); or c) coupling of two peptidic fragments by an amide bond, each of which contains at least one amino acid in protected or deprotected form and a peptide fragment containing a radical of formula (e) to (j) as defined in claim 1 , the peptide fragments being such that a protected or deprotected peptide having the sequence according to formula I above is obtained and, if necessary, removal of the protecting group (s) from a compound of formula I in protected form; or (d) for the production of a compound of formula I wherein X is a radical of formula (e) or (h) and A 1 is a radical of formula (k) (III) â € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒ- in claim 1, X 'is a radical of the formula (e1) or (h) as defined in claim 1, and Z is a leaving group, with a corresponding phenol, thiophenol or HX2 -pyridine or an acid of the formula HX- CO-Y, or a functional derivative thereof, or HX, -Y-, wherein X., Y. and Y. are as defined in claim 1, or e) for the production of a compound of the formula (I) wherein R 1, R 2, R 3 and R 4 are as defined in claim 1 and R 1 is hydrogen or C 1 -C 4 -alkyl. is a C1- aliphatic or alicyclic residue, the oxidation of a compound of the formula VH (V) wherein R1, A1 to A5, and R11 are as previously defined, and recovering a compound of the formula I or a pharmaceutically acceptable salt thereof. ester or amide thus obtained in free or salt form or in the form of a complex. sa. A compound according to any one of claims 1 to 6, characterized in that it is intended to be used as a pharmaceutical compound. 9a. A pharmaceutical composition comprising a compound of the formula (I) as defined in claim 1, or a physiologically hydrolyzable and acceptable ester or amide thereof, in free form or in a physiologically acceptable salt form, together with a pharmaceutically acceptable diluent or carrier -is acceptable. 12 10ê. A method for the treatment of disorders with an associated etiology or characterized by the excessive release of IL-β in a subject in need thereof, which method is characterized by administering to said subject an effective amount of a compound of the formula I as defined in claim 1 , a physiologically acceptable and hydrolyzable ester or amide thereof, or a pharmaceutically acceptable salt thereof. Lisbon, 2 November 1992 J. PEREIRA DA CRUZ Official Agent 4th Industrial Property RUA VICTOR COTOON, 10- A 3 «1200 USBOA